In FY2007, we have obtained sufficient structural constraints from solid state NMR and electron microscopy to develop candidate structural models for amylin fibrils. This progress is the result of considerable efforts to develop protocols for efficient chemical synthesis of full-length amylin with isotopic labeling of specific residues, as well as protocols for preparation of structurally homogeneous amylin fibrils. Successful, high-yield synthesis depends on the incorporation of Hmb-protected amino acids or pseudoproline dipeptides at certain sites during solid phase peptide synthesis, in order to disrupt secondary structure formation on the synthesis resin. Preparation of structurally homogeneous fibrils results from isolation of monomeric amylin fractions by gel filtration chromatography prior to fibril formation, and from the use of fibril seeds to self-propagate the desired morphology. The morphology that results from these protocols is a "striated ribbon", comprised of multiple 5-nm-wide protofilaments, laterally associated in a strictly parallel manner. Approximately ten amylin fibril samples, isotopically labeled at positions that span the entire amylin sequence, were prepared in this way. Measurements of mass-per-length by scanning transmission electron microscopy (collaboration with R.D. Leapman, DBEPS, NIBIB) indicate a protofilament mass-per-length of approximately 20 kD/nm, implying a structure that consists of two molecular layers in a cross-beta structural motif. Solid state NMR spectra reveal a single set of 13C chemical shifts, implying that all molecules are in the same structural environment, and consequently that the protofilament has two-fold symmetry. Secondary structure determined from chemical shifts and from quantitative measurements of backbone 15N-15N distances indicates the presence of two beta-strand segments, separated by a bend that allows these segments to come in contact with one another. Additional solid state NMR data indicate that the beta-sheets have an in-register parallel organization. Thus, the overall structure is a four-layered beta-sheet, comprised of two molecular layers. This very closely resembles the structure of Alzheimer's beta-amyloid fibrils with similar morphologies, which we have previously characterized (Petkova et al., Biochemistry 2006). However, whereas beta-amyloid fibrils are stabilized exclusively by hydrophobic contacts between beta-sheets, amylin fibrils appear to be stabilized by a combination of hydrophobic and polar interactions. [unreadable] [unreadable] This work is described in a full-length manuscript that has been published recently in Biochemistry. Additional work currently in progress includes: (1) Preparation of amylin microcrystals, which we discovered fortuitously to form under conditions closely related to conditions we use to prepare amylin fibrils. Dimensions of these microcrystals are too small to permit conventional x-ray crystallography, but we plan to attempt electron diffraction measurements in our own laboratory, and x-ray diffraction measurements on a new micro-focussed x-ray source at Argonne National Labs. If successful, a crystal structure of amylin will provide a wealth of new information about interactions that stabilize amylin fibrils; (2) Preparation of uniformly 15N,13C-labeled amylin by bacterial expression. The sharp 13C NMR lines observed in our solid state NMR spectra of synthetic, selectively-labeled amylin fibrils suggest that measurements on uniformly-labeled samples may have sufficiently high resolution to be interpretable. This will allow us to measure additional structural constraints that will pin down the high-resolution amylin fibril structure.